The present invention relates to attenuated strains of prokaryotic microorganisms, in particular Salmonella, transformed with nucleic acid encoding papillomavirus virus proteins, to compositions comprising these microorganisms, especially for use as vaccines, and to the medical uses of these strains. In a further aspect, the present invention provides a method of producing assembled papillomavirus virus like particles (VLPs).
Human papilloma virus (HPV) 16 is the major type of HPV which, in association with cofactors, can lead to cervical cancer (49). Studies on HPV have been hampered by the inability to propagate the virus in culture, the lack of animal models and the paucity of virions in clinical lesions. This has led to the development of alternative approaches of antigen production for immunological studies. The conformational dependency of neutralizing epitopes, as observed in experimental animal papillomavirus systems (8, 22) suggests that properly assembled HPV particles are critical for the induction and detection of clinically relevant immune reactivity.
The HPV capsids are formed by 72 pentameric capsomers of L1 proteins arranged on a T7 icosahedral lattice (15). Recently, a number of investigators have demonstrated the production of HPV capsids, i.e. virus like particles (VLP), by utilizing baculovirus, vaccinia virus or yeast expression systems (15, 22, 45, 48, 61). The potential of VLPs as subunit vaccines has been demonstrated using the cottontail rabbit papillomavirus (CRPV) (4), the canine oral papillomavirus (COPV) (57), and the HPVll models (45).
HPV16 infects through the genital mucosa, where benign proliferative lesions are confined. Protection against infection with such a pathogen could be provided by specific (anti-VLP) secretory immunoglobulins A (sIgA) or immunoglobulins G (IgG) in genital secretions. By analogy with existing animal models, HPV16 VLPs-specific antibodies in cervical secretions might help to prevent sexually transmitted infection by HPV16 in women. However, this cannot be formally proven in the absence of an experimental model for genital PV infection and other scenarios requiring cell-mediated immunity cannot be excluded.
Moreover, the mechanism underlying HPV infection is unclear. HPV may directly infect the basal cells of the stratified cervical epithelium at the occurrence of breaches. Alternatively, HPV infection could also occur either directly through Langerhans cells in intact epithelia or indirectly from an HPV-producing keratinocyte, and thus neutralizing antibodies will not be functional as shown for other viruses. This further adds to the difficulty in providing vaccines effective against HPV infection.
Immunosuppressed individuals are more prone to develop cervical carcinoma as compared to immunocompetent individuals, suggesting the possibility of using immunotherapy. Therapeutic vaccines (87) aimed to the treatment of established HPV infection or HPV associated premalignant and malignant lesions have been investigated during the last ten years (59). Evidence for HPV-antigen-directed immunotherapy against cervical cancer comes from the observations that experimental (13),(34),(83) and natural (82) PV-associated tumours can be controlled by immunization with E6 and E7 preparations. These studies suggested that CTL might be the most effective immunological effector mechanisms. E6 and E7 preparations consisted in either peptides (13), bacterially prepared fusion proteins (82), eukaryotic transfected cells (83) or recombinant vaccinia viruses (34).
Recently, chimeric VLPs carrying the 17kD E7 protein as a fusion with L2 have been shown to induce rejection of syngeneic tumour cells (84) engineered to express L1 and/or E7 ORF (i.e. C3 cells (13) and TC1 cells (85)). This data demonstrates the possibility of providing prophylactic and therapeutic effects in the same vaccine preparation.
Salmonella that are attenuated, yet invasive, have been proposed for the delivery of heterologous antigens to the mucosal and systemic immune systems (10). The antigen is delivered by the live Salmonella to mucosal inductive sites, where after priming, antigen-specific B and T cells migrate from the site of induction and mature into effector cells. The migrating IgA-expressing B cells home to different mucosal sites, including the genital tract, where they differentiate into IgA secreting plasma cells (32). Thus, oral or nasal immunization can provide protective antibodies in genital secretions. Recently, we and others have shown that mucosal immunization with recombinant Salmonella can elicit antibody responses in the genital mucosa of mice and humans (18, 37, 56).
In order to develop a prophylactic vaccine against HPV, we have expressed the major protein L1 of HPV16 in a PhoPc (35) attenuated strain of Salmonella typhimurium. Surprisingly, the inventors found for the first time that it is possible to assemble VLPs in a prokaryotic organism and that nasal immunization of mice with an HPV16-L1/Salmonella recombinant strain induces HPV16-specific conformationally dependent and neutralizing antibodies in serum and genital secretions. The experiments described herein also show that it is possible to assemble chimeric VLPs of a HPV protein and a fusion partner.
Accordingly, in a first aspect, the present invention provides an attenuated strain of a prokaryotic microorganism transformed with nucleic acid encoding papillomavirus virus major capsid protein wherein the protein assembles in the microorganism to form virus like particles (VLPs).
Thus, the present invention provides a way of producing properly assembled papillomavirus VLPs in an attenuated strain of a prokaryotic microorganism such as Salmonella so that they can be used as a vaccine to raise an immune response in a subject. Preferably, the VLPs are delivered to mucosal sites, having the advantage of generating the immune response to the papillomavirus VLPs at the locations where infection actually takes place, as well as at other mucosal surfaces.
The term xe2x80x9cpapillomavirusxe2x80x9d used herein covers both human and animal PVs. However, preferably, the papillomavirus is a human papillomavirus (HPV). About 70 different types of HPV have been cloned and characterized (denoted HPVL to HPV70 . . . ), and all have an 8 kb double stranded genome which encodes different early products and two late products L1 and L2, and are either epitheliotropic or mucosatropic. L1 is a major capsid protein and is relatively well conserved among the different HPV types. For a review of the HPV types and their nucleic and amino acid sequences, see Human Papillomaviruses, xe2x80x9cA Compilation and Analysis of Nucleic Acid and Amino Acid Sequencesxe2x80x9d, 1994, ed. Myers et al, Theoretical and Biophysics Group T-10, Los Alamos National Laboratory. Clinically, the most important HPV types are those that infect the anogenital tract, and that have high oncogenic risk and a high prevalence. This group includes HPV16, 18, 31 ,45 and 56, with HPV16 alone accounting for more than 50% of invasive cancer in the anogenital tract, as well as being the most prevalent single type of HPV.
The papillomavirus proteins correspond to wild type major capsid proteins (e.g. L1 and/or L2) or may be chimeras of all or part of a HPV protein and a fusion partner. The fusion partner may be any immunogenic protein against which specific CTL would be targeted. This protein may be an HPV protein (e.g. E7, E6 or E2 of any HPV type), a protein from another pathogen or any tumour specific antigen. In one embodiment, the HPV protein is L1 protein coexpressed with L2, with the fusion partner expressed so that it is linked to the L2 protein.
It has been shown that chimeric VLPs can elicit anti-tumour immunity against carrier and inserted proteins in HPV16 tumour models. Thus, chimeric VLPs which induce E7-specific CTLs aimed to the killing of already HPV infected cells or HPV-associated premalignant lesions. In this event, induction of CTLs to eliminate already HPV infected cells appears therefore an appealing complement to the induction of neutralizing antibodies, and chimeric VLPs have been shown to induce both functions.
Thus, in one embodiment of the invention, Salmonella strains able to induce neutralizing antibodies and CTLs by expressing chimeric VLPs could be therapeutic at least for early or pre-malignant HPV lesions in which the downregulation of MHC I or other factors observed in more advanced cancers has not yet occurred.
Preferably, the prokaryotic microorganism is an attenuated strain of Salmonella. However, alternatively other prokaryotic microorganisms such as attenuated strains of Escherichia coli, Shigella, Yersinia, Lactobacillus, Mycobacteria, Listeria or Vibrio could be used. Examples of suitable strains of microorganisms include Salmonella typhimurium, Salmonella typhi, Salmonella dublin, Salmonella enteretidis, Escherichia coli, Shigella flexeneri, Shigella sonnei, Vibrio cholera, and Mycobacterium bovis (BC6).
Attenuated Salmonella strains are one of the best characterized mucosal vaccine carriers. Recombinant Salmonella strains that are attenuated yet invasive have been used as oral vaccine vectors to carry protective epitopes of several pathogens into the mucosal associated lymphoid tissue thus inducing mucosal, systemic and CTL immune responses against both the carrier and the foreign antigens (58, 65, 67, 69, 75-77).
The currently licensed oral vaccine against typhoid fever S. typhi Ty21a (72) administered as a three-dose regimen of enteric-coated capsules (109 CFU/capsule) provided a 67% efficacy over a 3 year period. However, because the S. typhi Ty21a requires high and multiples doses in liquid formulation for higher efficacy, and its mutations are not yet all characterised (63, 64, 70, 71, 78), new attenuated Salmonella strains have recently been developed and tested in humans. These include nutritional auxotrophs in which pathways for biosynthesis of aromatic compounds have been interrupted (Aaro mutants). The xcex94aroA, xcex94purA mutants of S. typhi have been tested in human volunteers (32) and were shown to elicit specific cell-mediated immune responses but weak humoral responses. Other aro mutants (aroC and aroD) were insufficiently attenuated and caused fever and bacteremia (79). A double mutant xcex94aroC xcex94aroD Ty2 (CVD 908) was safe and elicited IgG antibodies against LPS in 80% of the immunized adult volunteers (73, 80). S. typhi mutants were also generated in which the adenylate cyclase (cya) and the cAMP receptor (crp) genes were deleted. These gene products are required for the transcription of many genes and operons that control transport processes, expression of fimbriae, flagella and some outer membrane proteins. One mutant X3927 (xcex94cya xcex94crp Ty2) was tested and shown to be immunogenic but some volunteers developed fever and vaccine bacteremia (79). Therefore, a novel strain, X4073, was constructed by deleting a third gene (cdt) responsible for colonization of deep tissue (66, 68, 74). This strain was administered to volunteers and proved to be completely safe at doses up to 5xc3x97108 CFU and generated a seroconversion in 80% of the volunteers (66).
Other attenuated Salmonella strains include mutants in a two-component regulatory system, the phoP/phoQ genes. These genes affect expression of a number of other genes and are responsive to phosphate levels and to enviromental conditions expected to be experienced by Salmonella residing within macrophages. One example of these mutants is the PhoPc strains used in the examples described below. Recently, a PhoP/PhoQ-deleted Salmonella typhi (ty800) has been shown to be safe and immunogenic in humans (81).
As mentioned above, the attenuated strain of the prokaryotic microorganism is transformed with a nucleic acid encoding one or more major papillomavirus capsid proteins. The inventors found for the first time that, when this nucleic acid is expressed in the microorganisms, the capsid proteins produced assemble correctly to form VLPs, making them especially suitable for the vaccination of subjects against papillomaviruses. Preferably, the major viral capsid protein is L1, optionally additionally including nucleic acid encoding L2 protein. As discussed above, the capsid protein may be linked to a fusion partner such as another antigen.
In a further aspect, the present invention provides a composition comprising one or more of above attenuated prokaryotic microorganisms, optionally in combination with a physiologically acceptable carrier. Preferably, the composition is a vaccine, especially a vaccine for mucosal immunization, e.g. for administration via the oral, rectal, nasal, vaginal or genital routes. Our earlier studies using recombinant Salmonella expressing hepatitis B virus antigen (18) showed that vaccination via any of these routes produces a sIgA response in the mucosal secretions at other sites. Advantageously, for prophylactic vaccination, the compositions comprises one or more strains of Salmonella expressing a plurality of different VLPs, e.g. VLPs from different papillomavirus types. This has the advantage of improving the protective effect of the vaccine to a range of challenges by the different papillomavirus types. For therapeutic vaccination, subsequent chimeric VLP constructs can comprise fusion products of various HPV type L1 capsids with the same L2 fusion partner.
In a further aspect, the present invention provides an attenuated strain of a prokaryotic microorganism described above for use as a medicament, especially as a vaccine.
In a further aspect, the present invention provides the use of an attenuated strain of a prokaryotic microorganism transformed with nucleic acid encoding papillomavirus virus major capsid protein, wherein the protein assembles in the microorganism to form virus like particles, in the preparation of a medicament for the prophylactic or therapeutic treatment of papillomavirus infection or anogenital cancer, especially cervical cancer.
Generally, the microorganisms or VLPs according to the present invention are provided in an isolated and/or purified form, i.e. substantially pure. This may include being in a composition where it represents at least about 90% active ingredient, more preferably at least about 95%, more preferably at least about 98%. Such a composition may, however, include inert carrier materials or other pharmaceutically and physiologicaly acceptable excipients. A composition according to the present invention may include in addition to the microorganisms or VLPs as disclosed, one or more other active ingredients for therapeutic use, such as an anti-tumour agent.
The compositions of the present invention are preferably given to an individual in a xe2x80x9cprophylactically effective amountxe2x80x9d or a xe2x80x9ctherapeutically effective amountxe2x80x9d (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practioners and other medical doctors.
A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.
Examples of techniques and protocols mentioned above can be found in Remington""s Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.
In a further aspect, the present invention provides a method for producing assembled papillomavirus virus like particles comprising culturing an attenuated strain of a prokaryotic microorganism transformed with nucleic acid encoding papillomavirus virus major capsid protein wherein the protein is expressed and assembles in the microorganism to form virus like particles. Preferably, the method additionally comprises the step of recovering the VLPs from the prokaryotic microorganism.
In a further aspect, the present invention provides the use of a papillomavirus VLP as obtainable by transforming an attenuated prokaryotic microorganism with nucleic acid encoding the VLPs and expressing the nucleic acid to produce assembled VLPs, in a diagnostic method. in one embodiment, present invention provides a method for detecting the presence of anti-papillomavirus antibodies in a sample from a subject comprising immobilizing the HPV VLPs on a solid support, exposing the support to the sample and detecting the presence of the antibodies, e.g. using ELISA.
Preferred embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying drawings.